On the Location of Stain in Mitochondria

1968 ◽  
Vol 26 ◽  
pp. 128-129
Author(s):  
G. B. Haydon ◽  
S. O. Smith
Keyword(s):  
Skeletal Muscle ◽  
Heavy Metal ◽  
Oxidative Phosphorylation ◽  
Focal Plane ◽  
Uranyl Acetate ◽  
Thick Section ◽  
Lead Citrate ◽  
Thin Sections ◽  
Thin Sectioning ◽  
Acetate Lead

The degree of matrical staining in isolated and tissue mitochondria has been shown to be a function of their state of oxidative-phosphorylation at the time of fixation (J. Histochem. Cytochem. 15:752, 1967). High resolution EM in conjunction with these studies revealed amplitude contrast images 10 AU and larger in diameter which are independent of the focal plane. These amplitude densities have been interpreted as individual aggregates of heavy metal stain (J. Cell Biol. 35:55A, 1967). Two approaches have been used in an effort to locate such stain aggregates.(1) Four micron epoxy sections of skeletal muscle, cardiac muscle, and liver were stained with uranyl acetate, lead citrate, or both, following the methods used to stain 500 AU sections for EM. After staining, the thick epon sections were re-embedded and thin sections cut perpendicular to the stained surface. Either or both of these metal stains penetrate 500 to 1000 AU into the thick section and give the tissue increased contrast similar to that due to staining after thin sectioning. The mitochondrial matrices are lightly stained indicating that they are not in a state of active oxidative-phosphorylation. Figure 1 shows such mitochondria at low magnification before (Figure 1a) and after (Figure lb) restaining with uranyl acetate and lead citrate. After restaining it is no longer possible to identify the original stained surface.

Morphometric comparison of uranyl acetate/lead citrate-stained and peroxidase-stained neutrophil granules

1990 ◽  
Vol 48 (3) ◽  
pp. 756-757
Author(s):  
Charles S. Gilbert ◽  
Richard T. Parmley ◽  
Joseph M. Kinkade
Keyword(s):  
Human Peripheral Blood ◽  
Uranyl Acetate ◽  
Buffy Coat ◽  
Significant Heterogeneity ◽  
Lead Citrate ◽  
Thin Sections ◽  
Low Viscosity ◽  
Physical Density ◽  
Acetate Lead ◽  
Blood Neutrophils

Although neutrophil granules can be broadly divided into peroxidase-positive and peroxidasenegative granules, recent studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. The present study was undertaken to evaluate this heterogeneity morphometrically. Human peripheral blood neutrophils were collected from normal volunteers in heparin or EDTA and fixed in 3% glutaraldehyde as a cell suspension or minced buffy coat. An aliquot was stained for myeloperoxidase using 3,3’-diaminobenzidine (DAB) as substrate. All samples were post fixed in 1% OSO4 and embedded in Spurr low viscosity medium. Additionally neutrophil granules isolated according to the method of Rice, et al. were similarly embedded. Thin sections of morphologic preparations were counterstained with methanolic uranyl acetate and aqueous lead citrate (UALC) (Fig. 1), whereas DAB stained specimens were counterstained with LC only (Fig. 2). Attempts to combine UALC and DAB staining in the same section obscured the density imparted by DAB. LC failed to impart significant density to DAB-negative granules in DAB-LC stained specimens resulting in an underestimation of this granule population.

A double staining technique for improved contrast of thin sections from Spurr-embedded tissue

1978 ◽  
Vol 56 (1) ◽  
pp. 129-132 ◽  
Author(s):  
D. F. Bray ◽  
E. B. Wagenaar
Keyword(s):  
Potassium Permanganate ◽  
Staining Technique ◽  
Uranyl Acetate ◽  
Staining Procedure ◽  
Lead Citrate ◽  
Double Staining ◽  
Thin Sections ◽  
Citrate Method ◽  
Acetate Lead

A double staining procedure using potassium permanganate and lead citrate is described. The technique, involving treatment of sections with a 1% solution of potassium permanganate followed by lead citrate staining, is shown to be more effective in producing contrast in Spurr-embedded tissue than the conventional uranyl acetate – lead citrate method.

Ultrastructural differences in the exosporium of the Sterne and Vollum strains of Bacillus anthracis

1968 ◽  
Vol 14 (12) ◽  
pp. 1297-1299 ◽  
Author(s):  
M. J. Kramer ◽  
Ivan L. Roth
Keyword(s):  
Electron Microscopy ◽  
Surface Layer ◽  
Bacillus Anthracis ◽  
Potassium Permanganate ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Single Application ◽  
Thin Sections ◽  
Acetate Lead ◽  
Different Strains

Spores from three different strains of Bacillus anthracis were examined by electron microscopy for the presence of a hair-like nap previously reported to be present on the exosporium of spores of the Sterne strain (avirulent). In addition to that strain, the Vollum strain (virulent) and a rough, avimlent variant of the Vollum were utilized in the current studies.Spores were fixed with Kellenberger's standard OsO4 fixative and embedded in Maraglas. Thin sections were poststained with various combinations of the following: potassium permanganate, uranyl acetate, lead citrate. The nap on the exosporium of spores of the Sterne strain was revealed most clearly when thin sections were poststained with all of the aforementioned stains. Post-staining by a single application of any of the three reagents resulted in a nap that was barely perceptible.The surface of the exosporium of spores from the Vollum strain and the rough, avirulent variant was found to be quite different from that of the Sterne strain. On the two former, the surface layer is approximately one-third as thick as the layer of hairs in the nap on the latter.

Lead aspartate: a new en bloc stain for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 34-35
Author(s):  
J.R. Walton
Keyword(s):  
Electron Microscopy ◽  
Calcium Ion ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc ◽  
Thin Sections ◽  
Lead Salts ◽  
Thin Sectioning ◽  
High Alkalinity

In electron microscopy, lead is the metal most widely used for enhancing specimen contrast. Lead citrate requires a pH of 12 to stain thin sections of epoxy-embedded material rapidly and intensively. However, this high alkalinity tends to leach out enzyme reaction products, making lead citrate unsuitable for many cytochemical studies. Substitution of the chelator aspartate for citrate allows staining to be carried out at pH 6 or 7 without apparent effect on cytochemical products. Moreover, due to the low, controlled level of free lead ions, contamination-free staining can be carried out en bloc, prior to dehydration and embedding. En bloc use of lead aspartate permits the grid-staining step to be bypassed, allowing samples to be examined immediately after thin-sectioning.Procedures. To prevent precipitation of lead salts, double- or glass-distilled H20 used in the stain and rinses should be boiled to drive off carbon dioxide and glassware should be carefully rinsed to remove any persisting traces of calcium ion.

Membranous alterations in the cytoplasm and nucleus in a primitive neuroectodermal tumor of the brain

1978 ◽  
Vol 36 (2) ◽  
pp. 628-629
Author(s):  
C. N. Sun ◽  
C. Araoz ◽  
H. J. White
Keyword(s):  
Nuclear Envelope ◽  
Tumor Cells ◽  
Osmium Tetroxide ◽  
Primitive Neuroectodermal Tumor ◽  
Uranyl Acetate ◽  
Neuroectodermal Tumor ◽  
Annulate Lamellae ◽  
Lead Citrate ◽  
Thin Sections ◽  
The Brain

The ultrastructure of a cerebral primitive neuroectodermal tumor has been reported previously. In the present case, we will present some unusual previously unreported membranous structures and alterations in the cytoplasm and nucleus of the tumor cells.Specimens were cut into small pieces about 1 mm3 and immediately fixed in 4% glutaraldehyde in phosphate buffer for two hours, then post-fixed in 1% buffered osmium tetroxide for one hour. After dehydration, tissues were embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate.In the cytoplasm of the tumor cells, we found paired cisternae (Fig. 1) and annulate lamellae (Fig. 2) noting that the annulate lamellae were sometimes associated with the outer nuclear envelope (Fig. 3). These membranous structures have been reported in other tumor cells. In our case, mitochondrial to nuclear envelope fusions were often noted (Fig. 4). Although this phenomenon was reported in an oncocytoma, their frequency in the present study is quite striking.

Unusual Intranuclear Structures in Chick Sympathetic Neurons

1967 ◽  
Vol 25 ◽  
pp. 188-189
Author(s):  
E. B. Masurovsky ◽  
H. H. Benitez ◽  
M. R. Murray
Keyword(s):  
Electron Microscope ◽  
Sympathetic Neurons ◽  
Uranyl Acetate ◽  
Sympathetic Ganglia ◽  
Lead Citrate ◽  
Thin Sections ◽  
Cns Neurons ◽  
Mammalian Cns

Recent light- and electron microscope studies concerned with the effects of D2O on the development of chick sympathetic ganglia in long-term, organized culture revealed the presence of rod-like fibrillar formations, and associated granulofibrillar bodies, in the nuclei of control and deuterated neurons. Similar fibrillar formations have been reported in the nuclei of certain mammalian CNS neurons; however, related granulofibrillar bodies have not been previously described. Both kinds of intranuclear structures are observed in cultures fixed either in veronal acetate-buffered 2%OsO4 (pH 7. 4), or in 3.5% glutaraldehyde followed by post-osmication. Thin sections from such Epon-embedded cultures were stained with ethanolic uranyl acetate and basic lead citrate for viewing in the electron microscope.

Fine Structure of Interdental Cells in the Mouse Cochlea

1970 ◽  
Vol 28 ◽  
pp. 140-141
Author(s):  
Roberta M. Bruck
Keyword(s):  
Fine Structure ◽  
Electron Microscope ◽  
Young Adult ◽  
Uranyl Acetate ◽  
Ground Substance ◽  
Tectorial Membrane ◽  
Lead Citrate ◽  
Unusual Structure ◽  
Thin Sections ◽  
Collagenous Fibers

An unusual structure in the cochlea is the spiral limbus; this periosteal tissue consists of stellate fibroblasts and collagenous fibers embedded in a translucent ground substance. The collagenous fibers are arranged in vertical columns (the auditory teeth of Haschke). Between the auditory teeth are interdental furrows in which the interdental cells are situated. These epithelial cells supposedly secrete the tectorial membrane.The fine structure of interdental cells in the rat was reported by Iurato (1962). Since the mouse appears to be different, a description of the fine structure of mouse interdental cells' is presented. Young adult C57BL/6J mice were perfused intervascularly with 1% paraformaldehyde/ 1.25% glutaraldehyde in .1M phosphate buffer (pH7.2-7.4). Intact cochlea were decalcified in .1M EDTA by the method of Baird (1967), postosmicated, dehydrated, and embedded in Araldite. Thin sections stained with uranyl acetate and lead citrate were examined in a Phillips EM-200 electron microscope.

Where is the prolamine located in rice endosperm?

1990 ◽  
Vol 48 (3) ◽  
pp. 696-697
Author(s):  
Hsin-Kan Wu ◽  
Mei-Chu Chung
Keyword(s):  
Storage Protein ◽  
Sodium Phosphate ◽  
Recent Experiment ◽  
Protein A ◽  
Uranyl Acetate ◽  
Protein Bodies ◽  
Lead Citrate ◽  
Thin Sections ◽  
Rice Endosperm ◽  
Ethanol Series

In one of our earlier papers (Wu et al. 1978), we suggested that glutelin is the major composition of the round storage protein bodies although they also contain relatively more prolamine than the angular one does. Immunochemical studies of Krishnan et al. (1986) later showed the presence of glutelin in the irregularly-shaped (angular) protein bodies while the prolamines were found in the round ones. Our recent experiment using protein A-gold technique found that prolamine is mainly deposited into the angular protein bodies.Small blocks (1 mm3) of 7 DAF (days after flowering) caryopsis of Orvza perennis were fixed with 3% paraformaldehyde and 3% glutaraldehyde in 0.1M sodium phosphate, pH7.4, dehydrated in a graded ethanol series and infiltrated with Spurr’s resin. Thin sections, after gold labeling, were stained with uranyl acetate and lead citrate. Rabbit antibodies were raised against purified prolamine. Protein A-gold sol complex was prepared based on the technique of Horisberger et al. (1977).

Recovery of the Venous Wall from Leukocyte Induced Injury

1975 ◽  
Author(s):  
G. J. Stewart ◽  
W. G. M. Ritchie ◽  
P. R. Lynch
Keyword(s):  
Smooth Muscle ◽  
Surface Membrane ◽  
Uranyl Acetate ◽  
Surgical Trauma ◽  
Luminal Surface ◽  
Lead Citrate ◽  
Jugular Veins ◽  
Acetate Lead ◽  
Partial Covering ◽  
Venous Wall

Surgical trauma to tissue adjacent to canine jugular veins combined with brief local stasis caused massive leukocyte invasion of venous walls. This resulted in the entrapment of pockets of leukocytes between the endothelium and basement membrane and subsequent detachment of patches of endothelium.By an hour smooth muscle showed a decrease in myofibrils and an increase in rough endoplasmic reticulum and collagen lost its ability to stain with uranyl acetate-lead citrate.By 24 hours smooth muscle cells had undergone various changes and formed a partial covering layer over the surface of denuded areas, often by extending long “arms” from a bulky, fibroblast-like “body”. Various stages of de-differentiation and re-differentiation were abundant. By 72 hours these cells had become thinner and resembled the “immature” endothelium of Florey and others. However, the sheet was still discontinuous. By 7 days and thereafter to 28 days the endothelium was continuous and typical of “immature” endothelium morphologically but the surface membrane stained intensely in some instances.At 24 hours there was a perivascular zone of edema with cell ghosts and amorphous debris which decreased thereafter. Collagen staining was again typical by 7 days.These observations indicate that the luminal surface of blood veins can be initially repaired by the rapid de-differentiation and re-differentiation of smooth muscle rather than waiting for the ingrowth of endothelial cells from the margins of the denuded areas.(Supported by N. I. H. Grants ≠ HL 14217 and HL 08886.)

An Electron Microscopic Investigation of the Pathogenesis of Hemorrhage Induced by Rattlesnake Venom

1974 ◽  
Vol 32 ◽  
pp. 56-57
Author(s):  
Charlotte L. Ownby ◽  
Robert A. Kainer ◽  
Anthony T. Tu
Keyword(s):  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Microscopic Investigation ◽  
Uranyl Acetate ◽  
Electron Microscopic Investigation ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Rattlesnake Venom ◽  
Diamondback Rattlesnake

One of the significant changes induced by the injection of rattlesnake (Crotalidae) venom is hemorrhage. Since crotaline antivenin does not prevent such local tissue damage, a more effective treatment of snakebite is needed. To aid in the development of such a treatment the pathogenesis of venom-induced hemorrhae was investigated.Swiss-Webster white mice were injected intramuscularly with Western diamondback rattlesnake (Crotalus atrox) venom. Two minutes after the injection, muscle tissue was obtained by bioosy from the thigh and fixed in 6% glutaraldehyde in Milloniq's phosphate buffer (DH 7.4, 2 hrs., 4°C). After post-fixation in 2% osmium tetroxide in Milloniq's phosphate buffer (pH 7.4, 1hr., 4°C) the tissue was dehydrated routinely in ethanol and embedded in Epon 812. The thin sections were stained with uranyl acetate in methanol and lead citrate then observed with either a Zeiss EM 9A or an Hitachi HS-8 electron microscope.

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